The Binding Interface between Bacillus stearothermophilus Ribosomal Protein S15 and its 5′-Translational Operator mRNA

The Bacillus stearothermophilus ribosomal protein S15 (BS15) binds a purine-rich three-helix junction motif in the central domain of 16 S ribosomal RNA (rRNA) as well as a translational operator located in the 5′-untranslated region (5′-UTR) of its cognate messenger RNA (mRNA). An in-frame fusion between the 5′-UTR of the BS15 gene and β-galactosidase (lacZ) was prepared, and tested for BS15-dependent translational repression of lacZ activity in Escherichia coli. The presence of BS15 in trans represses lacZ activity 24-fold. A series of detailed point mutations in BS15 were tested for their effects upon translational repression of lacZ activity. These point mutations demonstrated that the 5′-UTR-BS15 binding interface utilizes many of the same conserved amino acid residues implicated in the binding of BS15 to 16 S rRNA. The data demonstrate that the S15 protein can bind to an RNA target motif based primarily upon appropriate minor groove and sugar-phosphate backbone contacts, irrespective of the specific RNA sequence.